Gap junctions (GJ) are intercellular channels providing a pathway for transfer of ions and small molecules between adjacent cells of most tissues. Changes in intercellular gap junctional communication (IGJC) are believed to control various physiological tissue and cell functions as well as to be altered under pathological cell conditions. The degree of IGJC between cells depend on the number of GJ channels and their activity is a function of the state of phosphorylation of connexins (Cxs), the structural subunit of GJ channels. Protein phosphorylation has been proposed to control IGJC at several steps from gene expression to protein degradation, including translational and post- translational modification of Csx (i.e., phosphorylation of the assembled channel acting as a gating mechanism) and assembly into and removal from the plasma membrane. Several Cxs, including Cx32 and Cx43, contain sites for phosphorylation for more than one protein kinase. These consensus sites vary between Cxs. Using synthetic peptides with primary sequence homologous to that of C- terminal regions of Cxs we have identified in Cx32 a common site of phosphorylation for cAMP-dependent protein kinase and protein kinase (PKC). In addition, two putative sites of Cx43 phosphorylated by PKC were identified. We now propose to utilize molecular biological techniques to identify other seryl residues phosphorylated in Cx32 and Cx43 and elucidate the importance of their changes in state of phosphorylation. For this, IGJC-incompetent cell lines stably transfected with cDNAs encoding connexins whose phosphorylation sites have been changed by non-phosphorylatable amino acid residues using site-directed mutagenesis will be used. Results of these studies will be compared to those obtained in cell lines that naturally express Cx32 and/or 43 in order to dissect the effects due to phosphorylation of Cxs from indirect effects due to phosphorylation of proteins involved in different steps that control the degree of IGJC. These studies should contribute to our understanding of regulation of IGJC by protein phosphorylation under the effect of physiological and pathological stimuli.